Modulation of human V alpha 24(+)V beta 11(+) NKT cells by age, malignancy and conventional anticancer therapies

Immunotherapy strategies aimed at increasing human Valpha24(+)Vbeta11(+) natural killer T (NKT) cell numbers are currently a major focus. To provide further information towards the goal of NKT cell-based immunotherapy, we assessed the effects of age, cancer status and prior anticancer treatment on NKT cell numbers and their expansion capacity following alpha-galactosylceramide (alpha-GalCer) stimulation. The percentage and absolute number of peripheral blood NKT cells was assessed in 40 healthy donors and 109 solid cancer patients ( colorectal ( n = 33), breast ( n = 10), melanoma ( n = 17), lung ( n = 8), renal cell carcinoma ( n = 10), other cancers ( n = 31)). Responsiveness to alpha-GalCer stimulation was also assessed in 28 of the cancer patients and 37 of the healthy donors. Natural killer T cell numbers were significantly reduced in melanoma and breast cancer patients. While NKT numbers decreased with age in healthy donors, NKT cells were decreased in these cancer subgroups despite age and sex adjustments. Prior radiation treatment was shown to contribute to the observed reduction in melanoma patients. Although cancer patient NKT cells were significantly less responsive to alpha-GalCer stimulation, they remained capable of substantial expansion. Natural killer T cells are therefore modulated by age, malignancy and prior anticancer treatment; however, cancer patient NKT cells remain capable of responding to alpha-GalCer-based immenotherapies.

ISCOMATRIX (TM) adjuvant: An adjuvant suitable for use in anticancer vaccines

Human Papillomavirus type 16 (HPV16) E6 and E7 oncoproteins are associated with cervical cancer development and progression and can therefore be used as target antigens for cancer immunotherapy. In this study we evaluated the immunogenicity in mice, of different vaccine formulations using recombinant HPV16 derived E6E7 or E7GST fusion proteins. When co-administered with ISCOMATRIX(TM) adjuvant, these E6E7 proteins consistently induced E7 specific CTL, in vivo tumor protection, antibody and DTH responses. ISCOMATRIX(TM) adjuvant has been developed for use in the formulation of novel human vaccines and has been evaluated for safety and toxicity in human trials. A formulation containing aluminum hydroxide (Al(OH)(3)) gave a lesser degree of E7 specific antibody, and no local E7 specific CTL response but similar DTH and tumor protection. These findings demonstrate the potential of ISCOMATRIX(TM) adjuvant to stimulate both cellular and humoral immune responses to endogenously processed target antigens, and hence is the preferred adjuvant when CTL responses are desirable. (C) 2004 Elsevier Ltd. All rights reserved.

Exploiting novel cell cycle targets in the development of anticancer agents

In this review we provide a brief background on the cell cycle and then focus on two novel and emerging areas of cell cycle research that may prove to have significant relevance to the development of novel anticancer agents. In particular, we review the emerging evidence to suggest that histone deacetylase inhibitors may possess cancer cell-specific cytotoxicity due to their ability to target a novel G2/M checkpoint. We also review the recent literature supporting the proposition that inhibition of E2F activity in epithelial cancer cells may prove to be a useful differentiation therapy that operates via cell cycle-dependent and cell cycle-independent mechanisms.

Design, Synthesis, Potency and Cytoselectivity of Anticancer Agents Derived by Parallel Synthesis from alpha-Aminosuberic Acid

Chemotherapy in the last century was characterized by cytotoxic drugs that did not discriminate between cancerous and normal cell types and were consequently accompanied by toxic side effects that were often dose limiting. The ability of differentiating agents to selectively kill cancer cells or transform them to a nonproliferating or normal phenotype could lead to cell- and tissue-specific drugs without the side effects of current cancer chemotherapeutics. This may be possible for a new generation of histone deacetylase inhibitors derived from amino acids. Structure-activity relationships are now reported for 43 compounds derived from 2-aminosuberic acid that kill a range of cancer cells, 26 being potent cytotoxins against MM96L melanoma cells (IC50 20 nM-1 mu M), while 17 were between 5- and 60-fold more selective in killing MM96L melanoma cells versus normal (neonatal foreskin fibroblasts, NFF) cells. This represents a 10- to 100-fold increase in potency and up to a 10-fold higher selectivity over previously reported compounds derived from cysteine (J. Med. Chem. 2004, 47, 2984). Selectivity is also an underestimate, because the normal cells, NFF, are rarely all killed by the drugs that also induce selective blockade of the cell cycle for normal but not cancer cells. Selected compounds were tested against a panel of human cancer cell lines (melanomas, prostate, breast, ovarian, cervical, lung, and colon) and found to be both selective and potent cytotoxins (IC50 20 nM-1 mu M). Compounds in this class typically inhibit human histone deacetylases, as evidenced by hyperacetylation of histones in both normal and cancer cells, induce expression of p21, and differentiate surviving cancer cells to a nonproliferating phenotype. These compounds may be valuable leads for the development of new chemotherapeutic agents.

Synthesis of triplex-forming oligonucleotide conjugates of the anticancer drug temodal

Covalent attachment of the anticancer drugs temozolomide (Temodal) and mitozolomide to triplex-forming oligonucleotides (TFOs) is a potential way of targeting these alkylating agents to specific gene sequences to maximise site-selectivity. In this work, polypyrimidine TFO conjugates of both drugs were synthesised and targeted to duplex DNA in an attempt to effect site-specific alkylation of guanine residues. Concurrently, in an attempt to enhance the triple helix stability of TFOs at neutral pH, the thermal stabilities of triplexes formed from TFOs containing isoguanine, 2-O-benzyl- and 2-O-allyl-adenine were evaluated. A novel cleavage and deprotection procedure was developed which allowed for the solid phase synthesis of the base-sensitive TFO-drug conjugates using a recently developed silyl-linked controlled pore glass (SLCPG) support. Covalent attachment of either temozolomide or mitozolomide at the 5'-end of TFO conjugates caused no destabilisation of the triplexes studied. The synthesis of a phosphoramidite derivative of mitozolomide enabled direct incorporation of this reagent into a model sequence during DNA synthesis. After cleavage and deprotection of the TFO-drug conjugate, the 5'-end mitozolomide residue was found to have decomposed presumably as a result of ring-opening of the tetrazinone ring. The base-sensitive antibacterial and antitumour agent, metronidazole, was also successfully incorporated at the 5'-end of the oligonucleotide d(T8) using conventional methods. Two C2-substituted derivatives of 2'-deoxyadenosine containing 2-O-benzyl and 2-O-allyl groups were synthesised. Hydrogenolysis of the 2-O-benzyl analogue provided a useful route, amenable to scale-up, for the synthesis of the rare nucleoside 2'-deoxyisoguanosine (isoG). Both the 2-O-allyl and 2-O-benzyl derivatives were incorporated into TFO sequences using phosphoramidite methodology. Thermal melting experiments showed that the 2-O-allyl and 2-O-benzyl groups caused marked destabilisation of the triple helices studied, in contrast to hexose-DNA duplexes, where aralkyl substituents caused significant stabilisation of duplexes. TFOs containing isoG were synthesised by Pd(O)-catalysed deallylation of 2-0-allyl adenine residues. These sequences containing isoG, in its N3- or 02-H tautomeric form, formed triple helices which were equally as stable as those containing adenine.

Studies of cytotoxicity and anticancer mechanism of cytotoxic agents:cyclopentenones, cyclohexenomes, oxysterols and LR6M

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Pharmacogenomic studies of the anticancer and immunosuppressive thiopurines mercaptopurine and azathioprine

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT • 6-Mercaptopurine (6-MP) and azathioprine (AZA) are both inactive prodrugs that require intracellular activation into the active 6-thioguanine nucleotides (6-TGNs). • This metabolic process undergoes three different competitive pathways that are catalysed by three different enzymes; xanthine oxidase (XO), thiopurine methyltransferase (TPMT) and inosine triphosphatase (ITPA), all of which exhibit genetic polymorphisms. • Although the impact of genetic variation in the TPMT gene on treatment outcome and toxicity has been demonstrated, the role of other polymorphisms remains less well known. WHAT THIS STUDY ADDS • New information on the allelic variation of these three enzymes (XO, TPMT and ITPA) and their influence on 6-MP/AZA metabolism and toxicity. • Confirmation of the association of TPMT polymorphism with haematological toxicity. • Identified potential genetic characteristics that may contribute to higher risk of adverse events (such as ITPA IVS2+21A→C mutation). AIMS - To examine the allelic variation of three enzymes involved in 6-mercaptopurine/azathioprine (6-MP/AZA) metabolism and evaluate the influence of these polymorphisms on toxicity, haematological parameters and metabolite levels in patients with acute lymphoblastic leukaemia (ALL) or inflammatory bowel disease (IBD). METHODS - Clinical data and blood samples were collected from 19 ALL paediatric patients and 35 IBD patients who were receiving 6-MP/AZA therapy. All patients were screened for seven genetic polymorphisms in three enzymes involved in mercaptopurine metabolism [xanthine oxidase, inosine triphosphatase (C94→A and IVS2+21A→C) and thiopurine methyltransferase]. Erythrocyte and plasma metabolite concentrations were also determined. The associations between the various genotypes and myelotoxicity, haematological parameters and metabolite concentrations were determined. RESULTS - Thiopurine methyltransferase variant alleles were associated with a preferential metabolism away from 6-methylmercaptopurine nucleotides (P = 0.008 in ALL patients, P = 0.038 in IBD patients) favouring 6-thioguanine nucleotides (6-TGNs) (P = 0.021 in ALL patients). Interestingly, carriers of inosine triphosphatase IVS2+21A→C variants among ALL and IBD patients had significantly higher concentrations of the active cytotoxic metabolites, 6-TGNs (P = 0.008 in ALL patients, P = 0.047 in IBD patients). The study confirmed the association of thiopurine methyltransferase heterozygosity with leucopenia and neutropenia in ALL patients and reported a significant association between inosine triphosphatase IVS2+21A→C variants with thrombocytopenia (P = 0.012). CONCLUSIONS - Pharmacogenetic polymorphisms in the 6-MP pathway may help identify patients at risk for associated toxicities and may serve as a guide for dose individualization.

Anticancer effects of Curcuma C20-dialdehyde against colon and cervical cancer cell lines

Background: Recent attention on chemotherapeutic intervention against cancer has been focused on discovering and developing phytochemicals as anticancer agents with improved efficacy, low drug resistance and toxicity, low cost and limited adverse side effects. In this study, we investigated the effects of Curcuma C20-dialdehyde on growth, apoptosis and cell cycle arrest in colon and cervical cancer cell lines. Materials and Methods: Antiproliferative, apoptosis induction, and cell cycle arrest activities of Curcuma C20-dialdehyde were determined by WST cell proliferation assay, flow cytometric Alexa fluor 488-annexin V/propidium iodide (PI) staining and PI staining, respectively. Results: Curcuma C20 dialdehyde suppressed the proliferation of HCT116, HT29 and HeLa cells, with IC50 values of 65.4±1.74 μg/ml, 58.4±5.20 μg/ml and 72.0±0.03 μg/ml, respectively, with 72 h exposure. Flow cytometric analysis revealed that percentages of early apoptotic cells increased in a dose-dependent manner upon exposure to Curcuma C20-dialdehyde. Furthermore, exposure to lower concentrations of this compound significantly induced cell cycle arrest at G1 phase for both HCT116 and HT29 cells, while higher concentrations increased sub-G1 populations. However, the concentrations used in this study could not induce cell cycle arrest but rather induced apoptotic cell death in HeLa cells. Conclusions: Our findings suggest that the phytochemical Curcuma C20-dialdehyde may be a potential antineoplastic agent for colon and cervical cancer chemotherapy and/or chemoprevention. Further studies are needed to characterize the drug target or mode of action of the Curcuma C20-dialdehyde as an anticancer agent.

Cholecystokinin-1 receptor antagonists:5-hydroxy-5-aryl-pyrrol-2-ones as anticancer agents

A new class of 5-arylated 5-hydroxypyrrolones was derived from mucochloric acid in 2 synthetic steps and the chemical structure was confirmed additionally by X-ray analysis. Using a radiolabelled binding assay, potent CCK1 selective ligands were identified (CCK1: 12 nM) and the antagonism was confirmed by using isolated tissue preparations. A series of isobutyl derivatives displayed unsurmountable CCK antagonistic properties and in vitro excellent inhibition of proliferation was obtained in cholecystokinin related cancer cell lines in the nanomolar range. Finally, using xenograft studies in nude mice, two selected pyrrolone derivatives, X = H and X = F a fluorinated analogue (PNB-028), showed a strong inhibition of tumour growth in a chemo-resistant colon cancer-(MAC 16) and a human pancreatic cell line (MIAPACA) at 50 mg kg-1 by oral administration.

Anticancer activity of 4-N gemcitabine analogues

Gemcitabine (2', 2'-difluoro-2'-deoxycytidine or dFdC) has become a standard chemotherapeutic agent in the treatment of several cellular and solid tumor- related malignancies. Gemcitabine's anti-cancer activity has been attributed to its inhibitory effects on the cell's DNA synthetic machinery resulting in the induction of cell arrest and apoptosis. Despite its broad application, treatment capacity with this drug is limited due to complicated administration schedules stemming from low bioavailability and tumor resistance associated with its rampant intracellular enzymatic inactivation. The aim of this study is to characterize the anti-cancer activity of novel designed and synthesized gemcitabine analogues, that were modified with long alkyl chains at the 4-amino group of the cytosine ring. This study proposes the use of these alternative derivatives of gemcitabine that not only uphold current drug standards for potency, but additionally confer chemical stability against enzymatic inactivation. During screening conducted to identifY prospective gem-analogue candidates, I observed the potent anticancer properties ofthree 4-N modified compounds on MCF-7 breast adenocarcinoma cells. Experiments described here with these compounds referred to as LCO, LCAO, and Gvaldo, evaluate their cytotoxicity on MCF-7 cells at the concentrations of 25flM and 2.5flM, and assess their inhibitory effects on DNA synthesis and cell cycle progression using sulphorhodamine B and bromodeoxyuridine assays as well as flow cytometric analyses, respectively. Among the compounds tested, LCO was shown to be most active inhibitor of DNA synthesis (a=.05; p<.OOl) as reflected as a distinct GO/Gl versus S-phase arrest in the 25flM and 2.5flM treatments, respectively. Together, these experiments provide preliminary evidence for the clinical application of LCO-like gemcitabine derivatives as a novel treatment for breast cancer.

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues

Date of Acceptance: 28/07/2015 The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues

Date of Acceptance: 28/07/2015 The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).

Anticancer Properties of a Novel Class of Tetrafluorinated Thalidomide Analogues

Date of Acceptance: 28/07/2015 The authors thank Scott McMenemy for carrying out preliminary, early studies looking at effects of Gu compounds upon chicken embryology, as well as Charles D. Crowe, Jeffrey E. Roth, and Adam C. Rolt for critical comments on the article. fli1:EGFP zebrafish were obtained from the Zebrafish International Research Center (27). mpo:GFP zebrafish [also termed Tg(MPO:GFP)114] zebrafish were obtained from Dr. Stephen Renshaw, University of Sheffield (Sheffield, South Yorkshire, UK; ref. 29).